Renal cell carcinoma (RCC) encompasses 90% of kidney cancers and 3% of adult cancers. In the U.S. alone, more than 30,000 new cases of RCC are diagnosed and nearly 13,000 patients die from RCC annually. RCC is categorized into three distinct histologic subtypes of which clear cell (ccRCC) is the most common and lethal form, accounting for nearly 90% of RCC-related deaths. The majority of patients now diagnosed with ccRCC will present with organ-confined tumors seemingly amenable to surgical extirpation. Yet, almost half of these patients will experience metastatic relapse after surgery. Treatment options for metastatic ccRCC are limited both in terms of scope and efficacy. Thus, key advances to improve ccRCC patient care will necessarily include: 1) better methods to identify patients at high risk for metastatic relapse following surgery; 2) a better understanding of molecular mechanisms that drive ccRCC metastatic progression; and 3) rational, mechanism- targeted therapies to treat metastatic disease in ccRCC patients. Such improvements could emanate from the introduction of newer, more effective drugs or optimization of combination therapies. In this proposal, emphasis will be placed on the latter topic to gain a better understanding of key issues related to the immunobiology and immunotherapeutic treatment of advanced ccRCC. We have recently reported that ccRCC tumors can aberrantly express several relatively novel immune cell coregulatory ligands, namely B7-H1, B7-H3 and B7-H4. We have further shown that enhanced tumor expression of these B7-H ligands can predict aggressive ccRCC behavior including enhanced risk for cancer progression and cancer-related death. Collectively, these observations raise the distinct possibility that human tumors might employ B7-H ligands to disarm host antitumoral immunity in order to promote malignant progression. Thus, tumor-associated B7-H ligands will likely prove useful to refine prognostic algorithms to pinpoint high-risk patients who are most prone to ccRCC progression and death. However, whether tumor- associated B7-H ligands actually function as clinical inhibitors of cell-mediated antitumoral immunity to promote malignant progression remains to be determined. This proposal is singularly devoted to studies that, we believe, will directly improve clinical ccRCC patient care. Specifically, our studies will rigorously test the prognostic and immunotherapeutic potential of B7-H ligands aberrantly expressed by ccRCC. Specifically, we will address a most vital question: Do B7-H ligands within clinical tumors truly function to impair antitumoral immunity in cancer patients? Such findings will help to establish the merits (and limitations) of in vivo B7-H ligand blockade as a clinical immunotherapeutic approach to treat human forms of malignancy. Unifying Hypothesis for Specific Aims 1- 4: The overall hypothesis for this proposal is that the aggressive behavior of ccRCC tumors stems, at least in part, from the ability of tumors to elaborate an array of homologous but spatially distributed B7-H ligands that act in concert to undermine cell-mediated immunity in cancer patients. We further postulate that the most aggressive of ccRCC tumors concurrently express increased levels of other proteins that promote tumor cell proliferation and survival and, may also serve as antigenic moieties to lure immune cells into a perilous intratumoral environment. Thus, each aim of our proposal has been crafted to be free-standing, each addressing key clinical and scientific issues pertaining to B7-H ligands and moving from a macroscopic to microscopic understanding of B7-H ligands as immune- regulators and immunotherapeutic targets for clinical ccRCC. Specific Aim 1. Test if putative immunosuppressive, tumor-associated B7-H ligands (B7-H1, B7-H3, B7-H4, and PD-1) can collaborate with each other, as well as with other molecular markers that assist in fostering tumor cell proliferation and survival (IMP3, CAIX, Ki-67, and survivin) to improve outcome prediction for ccRCC patients. Specific Aim 2. Test whether ccRCC metastases are enriched for prognostic B7-H related molecules (B7-H1, B7-H3, B7-H4, and PD-1) as well as IMP3, Ki-67, and survivin when compared against patient-matched primary tumors from which metastases arise. Specific Aim 3. Test if soluble B7-H (sB7-H) ligands can be detected in the sera of ccRCC patients to improve strategies to monitor and, perhaps, immunotherapeutically treat ccRCC patients. Specific Aim 4. Test if tumor-associated B7-H ligands inhibit T and NK cell-mediated immune responses within clinical ccRCC specimens using in situ IHC analysis in combination with in vitro assays of immune cell function.